The long-range goal of this project is to understand the structure, function, and regulation of mammalian TRPC channels. TRP genes, originally identified as critical components of Drosophila phototransduction, encode a ubiquitous family of Ca2+-permeable cation channels that appear to play a fundamental role in cell signaling, cell growth, and cell death. TRP channels are divided into 3 major subgroups, TRPC, TRPV and TRPM. There are 7 mammalian TRPC proteins designated TRPC1-TRPC7. TRP channels are thought to be tetrameric, but the actual subunit composition remains unknown. Drosophila TRP channels are held in a large multimeric "signalplex" by the PDZ-containing scaffolding protein, INAD. We previously reported that immunophilin FKBP59, is a member of the Drosophila TRP signalplex. Recent studies suggest that TRPC1 C4, and C5 may also exist in a signalplex that includes FKBP52 and is tethered by a mammalian INAD-like protein, whereas TRPC3, C6, and C7 may form non-tethered heteromultimers, regulated by FKBP12. The specific aims of this project are to 1) define the native TRPC channel subunit composition, 2) identify and functionally characterize accessory proteins present in the TRPC channel signalplex, and 3) evaluate the role of immunophilins in regulation of TRPC channels. Towards these ends, multiple specific antibodies for each TRPC protein will be generated. The antibodies will be employed as immunohistochemical tools to evaluate tissue distribution and subcellular localization using confocal imaging techniques, and for immunoprecipitation experiments to isolate novel in vivo binding partners, i.e., "proteome walking". Following 2-D gel electrophoresis, novel interacting proteins will be identified by mass spectrometry. Channel function will be evaluated using patch-clamp and Ca 2+ imaging techniques, and structure-function relationships will be defined in heterologous expression systems using site-directed mutagenesis. High-throughput assays will be employed to evaluate protein-protein interactions, and to efficiently study regulation of TRPC channels by phospholipase C, protein kinases, and calmodulin, and to identify novel ligands that affect channel function and modify cellular signaling. Ultimately, these studies will provide greater insight into 1) the role TRPC channels in signal transduction, 2) their possible involvement in the development and/or progression of disease states, and 3) their potential use as molecular targets for novel therapeutic agents.